The present invention relates to a method for characterizing samples on the basis of intermediate statistical data.
The essence of a number of pharmacological, biological and chemical problems is to detect substances in a sample or to measure the interaction or reaction of these substances. In order to measure the substances in a sample more specifically, usually at least one of the reactants is radioactively or luminescently labelled. A convenient and sensitive type of labels are fluorescent labels.
Widely used methods to monitor interactions by fluorescence are the determination of changes in overall fluorescence intensity or in anisotropy of fluorescence. However, a number of side effects, such as surface binding or fluorescence from impurities, often lead to interpretation problems and artifacts. A second reason which has induced interest towards refined methods of analysis is the need to work with small amounts of a large number of samples in the field of high throughput screening and large capacity diagnostics.
New opportunities for assay development were opened when the technology for monitoring fluorescence from single fluorophore molecules became available. The first successful studies on fluorescence intensity fluctuations were performed by Magde, Elson and Webb (Biopolymers, Vol. 13, 29-61, 1974) who demonstrated the possibility to detect number fluctuations of fluorescent molecules and established a research field called fluorescence correlation spectroscopy (FCS). FCS was primarily developed as a method for determining chemical kinetic constants and diffusion coefficients. The experiment consists essentially in measuring the variation of the number of molecules of specific reactants in time in a defined open volume of solution. Microscopic fluctuations of the concentration of the reactant are detected as fluorescence intensity fluctuations from a small, open measurement volume. The measurement volume is defined by a focussed laser beam, which excites the fluorescence, and a pinhole in the image plane of the microscope collecting fluorescence. Intensity of fluorescence emission fluctuates in proportion with the changes in the number of fluorescent molecules as they diffuse into and out of the measurement volume and as they are created or eliminated by the chemical reactions. Technically, the direct outcome of an FCS experiment is the calculated autocorrelation function of the measured fluorescence intensity.
An important application of FCS is to determine concentrations of fluorescent species having different diffusion rates in a mixture. In order to separate the two terms corresponding to translation diffusion of two kinds of particles in the autocorrelation function of the fluorescence intensity, at least about a two-fold difference in diffusion time is needed, which corresponds generally to an eight-fold difference in the mass of the particles. Furthermore, if one succeeds in separating the two terms in the autocorrelation function of fluorescence intensity, it is yet not sufficient for determining the corresponding concentrations except if one knows the relative brightness of the two different types of particles.
The international patent application WO-A-98/16814 describes a method for characterizing samples by measuring a number of photon counts emitted, scattered and/or reflected by units in said sample in a repetitive mode per time interval of defined length, and determining a function of the number of photon counts per said time interval, characterized in that a function of specific brightness of said units is determined on basis of said function of the number of photon counts. This method can also be applied to study fluorescent samples. This special embodiment is the so called fluorescence intensity distribution analysis (FIDA). While FCS distinguishes between different species according to their diffusion time, FIDA distinguishes between them according to their specific brightness. Both FCS and FIDA rely on a single specific physical property. In principle, however, one-dimensional statistical functions obtained by FCS or FIDA can be used for determining distributions of particles in more than one specific physical property (e.g. distribution of particles according to both translational and rotational diffusion coefficient), but this option is limited and may be of low reliability.
The European patent application EP-A-0 359 681 discloses modulated dynamic light scattering methods which utilize time and space modulations of the incident or scattered light as well as modulations caused by random Brownian motions of the particles to measure particular properties.
A further method which utilizes fluorescence is Fluorescence Analysis in Cell Sorting (FACS). In FACS machines, the intensity of light emitted by a single particle is measured with a relatively high precision, and intensities corresponding to different wavelengths, scattering or polarization angles can be plotted simultaneously. U.S. Pat. No. 3,824,402 to Mullaney et al. discloses such a photometric apparatus and method for measuring light responsive characteristics of appropriately stained biological cells. At least two light responsive characteristics are measured and compared to eliminate spurious light induced noise. More particularly, light scattering produced by the cells and fluorescent light emitted by the cells in response to an incident light beam are detected. However, ordinary FACS cannot be applied in cases when the particles under study arrive and leave the measurement volume at random pathways, so that a lot of them do not pass through the centre of the focus. Also, the set of procedures used in FACS is not applicable in cases when light intensity corresponding to individual particles is so weak that the stochastical error of its measurement exceeds differences in intensities corresponding to different species of particles. Furthermore, FACS is applicable only at extremely low concentrations of particles, corresponding to much less than one particle per measurement volume.
One object of the invention is to increase the reliability of analysis of samples and reduce risk of misinterpretation of the measured data.
Another object of the invention is to considerably broaden the field of applicability of multidimensional analysis of samples.
The objects of the present invention are solved with the method having the features of characterizing samples which contain fluorescent molecules or particles, comprising the steps of:
a) monitoring intensity fluctuations of radiation emitted by the molecules or particles in at least one measurement volume by detecting sequences of photon counts by a single, two or more photon detectors,
b) determining from a probability function of at least two arguments, wherein at least one of the arguments is a number of photon counts n1 counted by detector 1 and another argument is a number of photon counts n2 counted by detector 2,
c) determining a distribution of molecules or particles as a function of at least two specific physical properties out of said intermediate statisical data.